Quantitative analysis of cryptic splicing associated with TDP-43 depletion.
Quantitative analysis of cryptic splicing associated with TDP-43 depletion.
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DOI：
10.1186/s12920-017-0274-1
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发表时间：
2017-05-26
影响因子：
2.7
通讯作者：
Plagnol V
中科院分区：
文献类型：
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作者：
Humphrey J;Emmett W;Fratta P;Isaacs AM;Plagnol V
可靠的外显子识别是前mRNA剪接成成熟mRNA的关键。TDP-43是一种RNA结合蛋白，其核丢失和细胞质聚集是肌萎缩侧索硬化和额颞叶痴呆（ALS/FTD）的标志性病理学。TDP-43缺失导致隐藏外显子异常包含在一系列转录物中，但其程度、与疾病发病机制的相关性以及是否由ALS/FTD中涉及的其他RNA结合蛋白引起尚不清楚。我们开发了一个分析管道来发现和量化隐藏的外显子包含，并将其应用于公开的人类和小鼠RNA测序数据。我们在TDP-43消耗数据集中检测到广泛的隐蔽剪接，但在另一种ALS/FTD相关蛋白FUS中几乎没有。对隐蔽外显子的序列基序和iCLIP分析表明它们被TDP-43结合。与hnRNP C耗竭中所见的隐蔽外显子不同，那些被TDP-43抑制的外显子不能与转座因子连接。隐藏的外显子保守性差，并且包含物压倒性地导致宿主转录物的无义介导的衰变，在差异表达分析中观察到转录物水平降低。73种不同RNA结合蛋白的RNA-蛋白相互作用数据显示，除了TDP-43，7个特异性结合TDP-43连接的隐蔽外显子。这表明TDP-43与其他剪接因子竞争结合隐蔽外显子，并且可以抑制隐蔽外显子包含。我们的定量分析管道证实了在TDP-43而不是FUS耗尽期间存在隐藏外显子，为ALS/FTD中RNA加工功能障碍的原因或后果提供了新的见解。本文的在线版本（doi：10.1186/s12920-017-0274-1）包含补充材料，可供授权用户使用。
Reliable exon recognition is key to the splicing of pre-mRNAs into mature mRNAs. TDP-43 is an RNA-binding protein whose nuclear loss and cytoplasmic aggregation are a hallmark pathology in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). TDP-43 depletion causes the aberrant inclusion of cryptic exons into a range of transcripts, but their extent, relevance to disease pathogenesis and whether they are caused by other RNA-binding proteins implicated in ALS/FTD are unknown. We developed an analysis pipeline to discover and quantify cryptic exon inclusion and applied it to publicly available human and murine RNA-sequencing data. We detected widespread cryptic splicing in TDP-43 depletion datasets but almost none in another ALS/FTD-linked protein FUS. Sequence motif and iCLIP analysis of cryptic exons demonstrated that they are bound by TDP-43. Unlike the cryptic exons seen in hnRNP C depletion, those repressed by TDP-43 cannot be linked to transposable elements. Cryptic exons are poorly conserved and inclusion overwhelmingly leads to nonsense-mediated decay of the host transcript, with reduced transcript levels observed in differential expression analysis. RNA-protein interaction data on 73 different RNA-binding proteins showed that, in addition to TDP-43, 7 specifically bind TDP-43 linked cryptic exons. This suggests that TDP-43 competes with other splicing factors for binding to cryptic exons and can repress cryptic exon inclusion. Our quantitative analysis pipeline confirms the presence of cryptic exons during the depletion of TDP-43 but not FUS providing new insight into to RNA-processing dysfunction as a cause or consequence in ALS/FTD. The online version of this article (doi:10.1186/s12920-017-0274-1) contains supplementary material, which is available to authorized users.